This study assessed the effect of magnifying endoscopy with narrow-band imaging (M-NBI) on the endoscopic differential diagnosis between intramucosal gastric carcinomas and adenomas with matched characteristics. Associations between magnified endoscopic findings and pathological high-grade cellular and architectural atypia were also investigated. In total, the records of 50 adenomas and 50 intramucosal well-differentiated adenocarcinomas matched by tumor size (≥ 20 mm or < 20 mm), shape (depression or non-depression), and color (red or non-red) were extracted. Fourteen endoscopists diagnosed adenoma or cancer in the 100 cases with conventional white light imaging (C-WLI), then did the same with C-WLI + M-NBI.The cancer diagnostic sensitivity, specificity, and accuracy were assessed. The sensitivity of C-WLI + M-NBI for cancer diagnosis was 79.9% compared to 71.6% with C-WLI (p < 0.001). There were no significant differences in specificity  and accuracy . High-grade cytological or architectural atypia was diagnosed more often with irregular microvascular pattern (IMVP) or microsurface pattern (IMSP), respectively, than the low-grade forms. In conclusion, IMVP and IMSP correlate with high-grade cytological and architectural atypia. M-NBI is useful in differentiating intramucosal carcinoma from adenoma and can reduce underdiagnosis of cancer. However, the 5-year relative survival rate of stage IA early gastric cancer is higher than 95%. Early gastric cancer can often be curatively treated with endoscopic resection. As endoscopic therapy is a stomach-preserving technique and minimally invasive, a higher standard of quality of life (QOL) can be preserved with endoscopic treatment compared to surgical treatment2. On the other hand, the 5-year relative survival rate of advanced gastric cancer with distal metastasis is lower than 10%4. Therefore, diagnosing gastric cancer at an early stage is very important for both improvements of prognosis and preservation of gastric function.Gastric cancer is the third leading cause of cancer death worldwide5. Early gastric cancer is often difficult to differentiate endoscopically from gastric adenoma, which is a benign epithelial tumor. In addition, lesions diagnosed as gastric adenoma by biopsy often result in a final diagnosis of cancer after endoscopic resection. There are several reports on the diagnostic accuracy of gastric adenoma and gastric cancer with conventional white light imaging (C-WLI). Kato et al. reported on endoscopic submucosal dissection (ESD) for 468 cases of gastrointestinal noninvasive neoplasia with preoperative Vienna classification category 3 or 4.1. They showed that the underdiagnosis rate after ESD was 44%, and that cancer was larger and more depressed than adenoma6. Cho et al. reported that in 236 cases of low-grade dysplasia (LGD)/adenoma on preoperative biopsy, 33.9% were high-grade dysplasia (HGD)/carcinoma after endoscopic resection and showed that the HGD/carcinoma are more depressed, red, and larger than LGD/adenoma7. In addition, Kim et al. reported that in 285 cases of LGD on preoperative biopsy, 16.1% were HGD/carcinoma, and showed that more than 2 cm, erythema, and depression were associated with upgraded histology after resection8. In this way, findings such as size, depression, and redness are known to be effective in differentiating gastric cancer from gastric adenoma with C-WLI. However, in the 285 cases Kim et al. mentioned above, regarding the findings for diagnosing cancer, the sensitivity and specificity for a size 2 cm or more were 37% and 87%, respectively; for redness, the sensitivity and specificity were 37% and 94%; and for depression, the sensitivity and specificity were 15% and 98%, respectively. Thus, diagnosis with C-WLI and preoperative biopsy have limitations in differentiating gastric cancer from adenoma. There is a risk of underdiagnosing cancer with diagnoses using only C-WLI or preoperative biopsy.In recent years, the detection rate of early gastric cancer has increased owing to the progress of endoscopic devices and diagnostic methods9. In this algorithm, gastric cancer is diagnosed when a demarcation line is present and an irregular microvascular pattern (IMVP) or irregular microsurface pattern (IMSP) can be confirmed with M-NBI. Although there are a few reports on the efficacy of M-NBI for differentiation between gastric cancer and gastric adenoma, it is not clear how effective M-NBI is for lesions that are difficult to differentiate with C-WLI12. Therefore, in this study, we investigated the effect of M-NBI in differentiating gastric cancer and adenoma matched by tumor size, shape, and color.In addition to C-WLI, since the diagnostic algorithm for early gastric cancer using M-NBI (MESDA-G) was published in 2016, diagnosis of early gastric cancer with M-NBI is used widely in Japan15. In a report on the Vienna classification, Western pathologists and Japanese pathologists compared 35 gastric tumors divided into five categories: reactive epithelium, low-grade adenoma/dysplasia, high-grade adenoma/dysplasia, suspected carcinoma, and definite carcinoma. Of the 29 cases diagnosed as definite cancer by Japanese pathologists, 3 were low-grade adenoma/dysplasia, 12 were high-grade adenoma/dysplasia and 10 were definite cancer by Western pathologists14. These results show Japanese pathologists diagnose cancer differently than Western pathologists. M-NBI may be able to observe the fine structure of the lesion in more detail than C-WLI. If cellular and architectural atypia can be detected endoscopically, it may be possible to diagnose cancer at an earlier stage.In pathological diagnosis, there is a difference in the diagnosis of cancer and dysplasia/adenoma between Europe and the United States and Japan. In Europe and the U.S., cancer is diagnosed by the disruption of the basal membrane in conjunction with the spread of cancer cells into the lamina propria. In contrast, in Japan, the diagnosis is based on cellular atypia and architectural atypia, irrespective of evidence of stromal invasionThe association between pathological cytological and architectural atypia with IMVP and IMSP in M-NBI findings was also investigated in this study.In the pre-matching patient characteristics, the frequencies of depression  and red color  were significantly higher in the cancer group than in the adenoma group. Tumor size was larger in the cancer group than in the adenoma group , but there was no significant difference between the proportions of ≥ 20 mm and < 20 mm (Table Helicobacter pylori (H. pylori) infection status, and mucosal atrophy between the cancer and adenoma groups . There were no significant differences in the sex or location of the lesion, ps Table .Table 2PIn pre-matching 691 cases (68 adenomas and 623 tub 1), when size ≥ 20 mm was diagnosed as cancer, the sensitivity, specificity, and accuracy were 18%, 91%, and 25%. Similarly, when depression was diagnosed as cancer, the sensitivity, specificity, and accuracy were 56%, 78%, and 58%. When redness was diagnosed as cancer, the sensitivity, specificity, and accuracy were 18%, 99%, and 26%, respectively.In 100 post-matched cases, the mean values of sensitivity, specificity, accuracy, and the positive predictive value (PPV) and negative predictive value (NPV) with C-WLI were 71.6%, 40.1%, 55.9%, 54.6% and 60.1%, respectively. Similarly, the mean values of sensitivity, specificity, accuracy, and PPV and NPV with C-WLI + M-NBI were 79.9%, 36.3%, 58.1%, 56.0% and 65.2%, respectively , lesion factors , or endoscopist factor (years of M-NBI experience) were associated with the accuracy of diagnosis using C-WLI + M-NBI  were diagnosed with invasive cancer from LGD at a mean 48 months of follow-up20. Okamoto et al. reported that 129 of 376 patients (34%) with gastric adenoma were diagnosed as cancer at the 5-year follow-up, and metachronous gastric cancer was diagnosed in 1.5% of the patients annually21. Considering the diagnostic limitations of M-NBI, regular follow-up may be necessary even for patients with endoscopically diagnosed adenoma.Regarding the long-term follow-up of LGD, Yamada et al. reported that only 1 of 38 cases (3%) were upgraded from category 3 (low-grade adenoma/dysplasia) to category 4  by biopsy at a median follow-up of 4.7 years20. Sakurai et al. reported that subsequent ESD demonstrated submucosal infiltration and/or venous invasion in 3.8% and 1.3% in HGD cases22. The pathological diagnosis based on cellular and architectural atypia in Japan may be earlier than by pathologists in the in the West. In this study, IMVP and IMSP were shown to be associated with these cellular and architectural atypia, and attention to the findings of IMVP and IMSP may allow a somewhat earlier diagnosis. This study proves the concept that endoscopic diagnosis can provide a diagnosis that is highly correlated to pathology. In recent years, endoscopes capable of 520 × magnification and evaluation of cell nuclei have been released. However, these endoscopes are still not as versatile as the magnifying endoscope used in this study due to the need for staining and observation techniques. In the future, with further advances in endoscopy, magnifying endoscopy will be closer to the pathological diagnosis, and optical biopsy may become the golden standard for clinical decisions in the future.In addition, we were able to show that IMVP and IMSP in M-NBI were associated with pathological high-grade cellular atypia and high-grade architectural atypia respectively. In a long-term follow-up of patients with gastric noninvasive neoplasia for more than 12 months, there was a significant difference in the number of patients diagnosed with invasive carcinoma during the follow-up period: LGD was 9% vs. HGD was 69%H. pylori infection.This study has several limitations. It was a single-center retrospective study with a limited number of cases. In this study, all images in which the target lesion could be at least partially visualized were extracted to reduce selection bias in endoscopic images, but because diagnosis is made retrospectively with a limited number of images, less information was available than in real-time diagnosis, which may affect the diagnosis. Eradication history was not considered for In addition, since gastric adenomas with atypical endoscopic findings are more likely to undergo resection, there is a possibility of selection bias, especially for gastric adenoma cases. A prospective, multicenter study to confirm the results of this study is desirable.In conclusion, M-NBI is useful in differentiating gastric intramucosal carcinoma from gastric adenoma and can reduce the underdiagnosis of cancer and resection should be considered when there are M-NBI findings suggestive of cancer. This study also shows IMVP and IMSP observed with M-NBI were associated with pathological high-grade cytological and architectural atypia.23. Vienna classification Category 3 (low-grade dysplasia) corresponds to adenoma in the Japanese classification, and Category 4 and 5 (high-grade neoplasia) to carcinoma.The macroscopic type of early gastric cancer was further classified into 2 groups: depressed (type 0–IIc or type 0–III) or non-depressed (other than type 0–IIc or type 0–III).This is a single center retrospective analysis on endoscopic diagnosis of gastric adenoma and gastric cancer. All resected lesions were histologically diagnosed by pathologists specializing in gastroenterology. Location, macroscopic type, histologic type, and invasion depth were defined according to the Japanese classification of gastric carcinoma by the Japanese Gastric Cancer AssociationPathological cellular and architectural atypia were reevaluated by two pathologists specializing in gastroenterology.A comprehensive retrospective analysis of each patient’s medical record was performed after approval by the Research Ethics Committee, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo (No. 2058). Written informed consent was waived by the by the Research Ethics Committee, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo owing to the retrospective design of this study. The present study was performed in accordance with the Declaration of Helsinki.H. pylori infection status was assessed with either H. pylori antibody, urea breath test, stool antigen test, or rapid urease test regardless of H. pylori eradication history. Mucosal atrophy was divided into the closed type and open type by the Kimura-Takemoto classification24.In total, 691 cases with intramucosal well differentiated adenocarcinoma (tub1) and gastric adenoma diagnosed after endoscopic resection at the University of Tokyo from April 2011 to July 2018 were retrospectively reviewed. The following were excluded: cases without M-NBI images and cases with familial adenomatous polyposis (FAP). After performing 1 to 1 matching by size (≥ 20 mm or < 20 mm), shape (depressed or non-depressed) and color (red or non-red), a total of 50 cases of adenomas and 50 cases of cancers were included for analysis Fig. . If therPatients were prepared with 10,000 units of Pronase and 1 g of sodium bicarbonate, and 40–80 mg of dimethicone before the endoscopic examination to remove mucus. A magnifying upper gastrointestinal endoscope  and electronic endoscopy system  were used. A video processor was set up as follows: color mode 1 and structure stressed A mode: level 5 or color mode 1 and structure stressed B mode: level 6 during conventional white light endoscopy; and color mode 1 and structure stressed B mode: level 8 during magnifying endoscopy with NBI. A soft black hood was mounted at the tip of the endoscope to obtain a stable magnification image.For each of the 50 cancers and 50 adenomas, endoscopic images of the target lesion were extracted from the medical records. All images in which the target lesion could be at least partially visualized were extracted, resulting in a total of at least two images each of C-WLI and M-NBI, including close-up and distant views when possible. Fourteen endoscopists participated in this study. Seven were experienced endoscopists (experts) with more than three years of M-NBI experience and the remaining seven were endoscopic fellows (non-experts) with less than three years of M-NBI experience. All endoscopists performed a differential diagnosis of either gastric cancer or adenoma with only C-WLI. Subsequently, they performed quantitative diagnosis of microvascular and microsurface patterns, and differential diagnosis of adenoma or cancer after observing the C-WLI and corresponding M-NBI images Fig. . For anaFor these 100 cases, the sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV) of each endoscopist were determined. The mean of sensitivity, specificity, accuracy, PPV and NPV of 14 endoscopists with C-WLI was defined as the diagnosis result with C-WLI. In the same way, the mean values of sensitivity, specificity, accuracy, PPV and NPV of 14 endoscopists with C-WLI + M-NBI were determined, and the results of C-WLI and C-WLI + M-NBI were compared. The percentage of an accurate diagnosis for each case was defined as the percentage of endoscopists whose endoscopic diagnosis was consistent with the pathological diagnosis. In addition, the association between diagnostic accuracy and background factors was also investigated.14. For M-NBI findings, the number of endoscopists who judged the presence of IMVP and IMSP in each case was determined. Next, the association of pathological high-grade cellular atypia and high-grade architectural atypia with the ratio of endoscopic diagnosis of IMVP and IMSP was evaluated.One hundred gastric tumors were reevaluated for cytological and architectural atypia by two expert gastrointestinal pathologist (H.A. and T.U.). Cellular atypia was divided into two categories, high-grade and low-grade, based on cytological changes . Architectural atypia was also divided into two categories, high-grade and low-grade, based on architectural changes 25. Statistical analysis was performed using JMP Pro version 16  and R statistical package .The primary aim of this study was to assess the efficacy of M-NBI in differentiating gastric cancer from gastric adenoma with matched characteristics. Categorical variables were analyzed using Pearson’s chi-squared test or Fisher’s exact test, and continuous variables were analyzed using Student’s t-test or the Wilcoxon rank-sum test, as appropriate. Paired t-test or Wilcoxon signed-rank test was used to compare diagnostic rate, as appropriate. Two-sided p-values < 0.05 were considered statistically significant. As a measure of inter-endoscopist agreement, Fleiss’s kappa was used. Values of kappa near zero indicate chance agreement only, while values near the maximum of 1 indicate perfect agreement. Kappa is judged as providing poor agreement if k ≤ 0.20; fair agreement if 0.21 ≤ k ≤ 0 0.40; moderate agreement if 0.41 ≤ k ≤ 0.60; substantial agreement if 0.61 ≤ k ≤ 0.80; and good agreement if k > 0.80


This study proposes a high-temperature superconducting (HTS) bandpass filter with a continuously tunable bandwidth and center frequency. The proposed filter combines several gallium arsenide varactors and a dual-mode resonator (DMR). The even and odd modes of the DMR can be tuned simultaneously using a single bias voltage. The capacitive value of varactors in the circuit is tuned continuously under continuous voltage and frequency tunability. External couplings and the interstage can be realized using an interdigital coupling structure; a fixed capacitor is added to the feeder to improve its coupling strength. A low-insertion loss within the band is obtained using HTS technology. Additionally, the proposed filter is etched on a 0.5 mm-thick MgO substrate and combined with YBCO thin films for demonstration. For the as-fabricated device, the tuning frequency range of 1.22~1.34 GHz was 9.4%; the 3-dB fractional bandwidth was 12.95~17.39%, and the insertion loss was 2.28~3.59 dB. The simulation and experimental measurement results were highly consistent. Filters are crucial in modern wireless communication systems (WCSs). Over the past decades, various filters have been proposed and thoroughly investigated ,2,3. RecIn this study, a four-pole HTS-tunable filter consists of two DMRs, which can be applied to WCSs. The resonator can be tuned simultaneously using a single bias voltage (SBV). Meanwhile, external couplings and interstage can be realized using an interdigital coupling structure, and a fixed capacitor is added to the feeder to improve the coupling strength. In addition, the HTS-tunable filter is simulated by Sonnet. The tunable filter was fabricated on a 0.5 mm-thick MgO substrate with YBCO films, and the relative dielectric constant (RDC) of the substrate was 9.8. The circuit dimension was 21.6 mm × 13 mm, and it was mounted on a metal carrier and fabricated on a metal shield box. By changing the SBV, the center frequency variable range of the HTS-tunable filter is 1.22~1.34 GHz. The 3-dB fractional bandwidth increased from 12.95% to 17.39%. The results of simulations and experimental measurements are highly consistent.vC. Additionally, the grounding of the short transect is realized using superconducting grounding pads (GPs).The proposed resonator is shown in The resonance characteristics of this resonator can be investigated using the even–odd mode method owing to symmetrical characteristics. The influence of the finger line on the open end is ignored for simplicity of analysis. The odd- and even-mode equivalent circuits are shown in ineY can be deduced as1 = Yk1/Y2, k2 = Y2/Y4, k3 = Y1/Y4, 1p = tanθ2 + tanθ3, p2 = k1 tanθ3 + k3tanθ4. The odd-mode input admittance inoY shown in k1 = Y1/Y2. When inoY = 0 and ineY = 0 are applied, the odd- and even-mode resonance conditions can be obtained.For the even mode of the resonator shown in vC influences the even- and odd-mode frequencies. Therefore, the odd- and even-mode frequencies can be adjusted using a variable capacitor only. This simplifies the adjustment of the filter frequency. Additionally, the SCS  influences the even-mode frequency only, as shown in As shown in o and fe represent odd and even mode frequencies, respectively. As shown in o remains unchanged with a variation in W and L, as long as the other variables remain constant. However, fe decreases as L increases, and fe increases as W increases. In other words, the odd- and even-mode frequencies can be controlled separately, which can be easily tuned to the desired frequency band. Therefore, the bandwidth of the filter can be easily controlled. This is consistent with findings from previous studies.The frequency response of the resonator is analyzed under weak coupling conditions by using Sonnet. The modal resonance characteristics at different microstrip line lengths of the resonator (L and W) are shown in vC at each end of the OCS. Interdigital coupling is employed for inter-resonator coupling. The finger length is adjusted to achieve the designated coupling. The input/output feedlines, with fixed capacitors C, are coupled to the DMR to achieve the designated external coupling. Additionally, four transmission zeros are introduced into the upper stopband and one transmission zero into the lower stopband to improve out-of-band suppression. Meanwhile, the two GPs (upper and lower) provide radio frequency (RF) and DC grounding, wherein RF signals are shorted using the DC blocking capacitors (Cd), whereas DC signals are blocked using the Cd. The DC bias pads are connected to the SCSs of the DMRs using a high-impedance line to avoid leakage of RF signals.The proposed HTS-tunable filter with four poles is shown in 2, whereas the lower HTS GP has a size of 21.6 × 2 mm2 with negligible impact on the RF performance. The soldering bias pad, with dimensions of 1 × 1 mm2, is connected to the resonator. This connection is achieved using a high-impedance line with a width of 0.2 mm.The grounding technique is essential for the design of an HTS-tunable filter. The resonant frequency is influenced by the HTS GP, which is influenced by its width. The upper HTS GP shown in vC decreases as the applied bias voltage (BV) increases. Thus, the center frequency of the filter increases as BV increases. Once the initial resonator structure is determined, the coupling control mechanism is investigated to satisfy the requirements of the design parameters  for the particular specifications in the passband.k at the resonant frequencies may be calculated as1f and f2 refer to lower and upper coupling resonant frequencies, respectively.The coupling configuration proposed is shown in 3 = 1.48 mm. k, a function of g and L3, and vC are obtained by tuning the varactor's value with the others maintained constant. The results are shown in The initial distances, in this case, are g = 1.92 mm and LvC = 0.2~0.9 pF under other variables is shown in vC remains constant, the k of the resonators decreases with increasing g. As shown in vC remains constant, the k of the resonators increases with increasing L3.The coupling coefficient when 3 significantly influences k. Herein, L3 is determined, and k is then fine-tuned by g. Therefore, the value of k can satisfy the desired value by selecting appropriate parameters.Compared with g, Lf0 refers to the resonance frequency, and ΔfdB3 refers to the 3 dB bandwidth. As shown in vC has a suitable eQ ranging from 0.2 to 0.9 pF. The external coupling of the filter can be easily adjusted by changing the capacitance value of C. By changing the capacitance value of C, the value of the external coupling can be appropriately changed, so that the filter can work normally under different center frequencies.A pair of fixed capacitors C is connected to the input and output matching networks to adjust the external coupling. The external quality factors can be calculated as follows :(6)Qe=f0The four-pole HTS-tunable filter, whose relative bandwidth increased with increasing tunable frequencies, was simulated using the Sonnet. The circuit dimensions shown in vC to the circuit was achieved using the power supply via feed-through capacitors. Additionally, a fixed capacitor C was placed between the feeders to tune eQ. The varactor capacitances were determined using a pre-fabricated HTS-tunable filter that ranged from 0.9 to 0.2 pF, whereas the reverse BV ranged from 1~13 V.Particularly, the tunable filter was fabricated on a 0.5 mm-thick MgO substrate with YBCO films, and the RDC of the substrate was 9.8. The circuit dimension was 21.6 mm × 13 mm, and it was mounted on a metal carrier and fabricated on a metal shield box. Varactors (M/A-COM MA46H120), 18 pF fixed capacitors, and 100 nF DC blocking capacitors, characterized by miniaturization and high Q-factor, were mounted to the circuit using silver epoxy glue. An image of the as-prepared four-pole filter is shown in The filter was cooled to 77 K. The results of the experimental measurements are shown in A four-pole compacted HTS-tunable filter was implemented using a DMR. Four transmission zeros were generated in the upper stopband, and one transmission zero was generated in the lower stopband using a feedline structure. The filter frequency was tuned by applying an SBV to four varactors. The rationality of the circuit design was validated through simulations and experiments, and the HTS-tunable filter, whose bandwidth increased with tunable frequencies, was fabricated. The proposed filter had a high filtering performance and excellent suitability for multi-service communication applications.


Curcumin possesses beneficial biological functions, namely anti-inflammation and anti-diabetic functions. However, due to its low solubility and crystallinity, its applications are limited. In this work, curcumin was encapsulated in casein micelles in order to form curcumin-casein nanoparticles by ultrasound treatment (5 min). The ultrasound treatment induced the entry of the hydrophobic groups to the inner micelles and the polar sulfydryl groups to the surface of the micelles in order to form compact curcumin-casein nanoparticles of an appropriate size (100–120 nm) for cellular endocytosis. The product exhibited excellent stability during 8 months of cold storage, 6 days at room temperature, and 2 days at body temperature. Advanced in vitro experiments demonstrated that curcumin-casein nanoparticles displayed significantly greater inhibitory activity against the proliferation and proinflammatory cytokines of human fibroblast-like synoviocyte-osteo arthritis (HFLS-OA) cells and HFLS-rheumatoid (RA) cells than native curcumin due to better cellular uptake as a result of the low crystallinity and the appropriate nano-size of the nano-form. The results provide a reference for the use of ultrasound treatment to encapsulate other drug molecules and curcumin-casein nanoparticles as potential treatment for arthritis. Curcumin is the primary polyphenol in the curcuminoid family of turmeric, a prominent ginger family spice. Curcumin has a wide range of beneficial biological activities, namely anti-inflammatory, anti-diabetic, antibacterial, and anti-cancer properties, aside from its traditional use as an antioxidant and a food coloring. However, curcumin has drawbacks, such as a short shelf life due to its low stability, poor bioavailability due to its low water solubility and poor absorption, quick systemic elimination, and quick metabolism. Nanoencapsulation is an emerging strategy that is employed in order to resolve these drawbacks. For the loading of curcumin, several delivery systems have been employed, including nature-inspired, polymer- and biopolymer-based, lipid-based, surfactant-based, and special equipment-based methods .Curcumin that is encapsulated in nano-emulsions has higher dispersibility than raw curcumin . LiposomCaseins, the main protein constituent of bovine milk, are a commercially accessible food-grade additive in beverages and food . CaseinsCasein nanoparticles have also been encapsulated with curcumin. Sahu et al.  encapsulUltrasound-assisted or sonication strategies have emerged as possible candidates in numerous processes/industries. However, the heat generated by an ultrasound affects the properties of the materials. Sylia et al.  discusseCurcumin (>99.0%) was acquired by Taizhou Crene Biotechnology Co., Ltd., . CCK-8 and sodium casein (90%) were acquired from Sigma–Aldrich Corp. . Tumor necrosis factor α (TNF-α) and the enzyme-linked immunosorbent assay (ELISA) kits for human vascular endothelial growth factor, human TNF-α, human interleukin-2 like (IL-2L), human interleukin-6 (IL-6), and human matrix metalloproteinase 1 (MMP-1) were acquired from Sigma–Aldrich . The other reagents utilized were high-performance liquid chromatography (HPLC), biological, and analytical grades.Human fibroblast-like synoviocyte–rheumatoid arthritis cells (HFLS-RA) and human fibroblast-like synoviocyte–osteoarthritis cells (HFLS-OA) were acquired from Shanghai Cells Bank, Institute of Life Sciences, Chinese Academy of Sciences .Sodium caseinate was fully dissolved in water and four solutions of 10, 20, 30, and 40 mg/mL concentrations were prepared. Each caseinate solution (20 mL) was placed in a 50 mL tube and ultrasonicated  using a SCIENTZ-950E Ultrasonic processor . The tube was placed in an ice bath in order to control the temperature during ultrasonication. After the ultrasonication, the caseinate dispersion was filtered using a 460 nm film for the advanced measurement of size, zeta potential, polydispersion index (PDI), surface free sulfydryl and total free sulfydryl, and the surface hydrophobicity of the casein micelles.At 25 ± 0.1 °C and a scattering angle of 90°, the size of the nanoparticles was studied utilizing a zeta potential measurement . The volume measurement was used to assess particle size distribution, which determines the proportional percentage of particles in each size category based on the volume they cover. Laser Doppler velocimetry (LDV) was used in order to determine the zeta potential utilizing the same Zetasizer Nano-ZSE analyzer .412 denotes the absorbance at 412 nm, C denotes the casein micelle dispersion content (mg/mL), the factor 73.53 was obtained from 106/(1.36 × 104), and 1.36 × 104 denotes the molar Ellman absorptivity constant.The content of the free sulfhydryl groups (SH) of the casein micelle samples containing free SH was determined using Ellman’s reagent , DTNB) . Brieflyw/v) casein micelle dispersions were produced using phosphate buffer . An amount of 0.6 mL of 8-anilino-1-naphthalene sulfonic acid  was dissolved in 10 mL of all the dilutions and was allowed to react for 15 min. An F-7000 fluorescence detector  was utilized in order to analyze the fluorescence of each sample. The analysis was conducted with excitation and emission at 390 nm and 480 nm, respectively. The initial slope of the relative fluorescent intensity (RFI) versus the protein content of the serial dilutions was used to compute the solutions’ surface hydrophobicity index (SHI). RFI was described by RFI = (F − F0)/F0, where F0 is the reading of the ANS solution without casein and F is the fluorescence reading of the protein–ANS conjugate.The surface hydrophobicity of the casein micelle samples was assessed based on the approach used by Wang et al. . In ordew/w). The mixture was ultrasonicated for 2.5, 5, 7.5, and 10 min. The denatured protein was eliminated by filtering with a 460 nm film after ultrasonication. Finally, a curcumin–casein nanoparticle (Cur-CS-NPs) dispersion was obtained. The curcumin amount in the Cur-CS-NPs dispersion was fully regarded as encapsulated curcumin because the non-encapsulated curcumin was removed by filtration.Sodium caseinate solutions of 10, 20, 30, and 40 mg/mL concentrations were obtained by completely dissolving the appropriate amounts of sodium caseinate in water, and curcumin solution with a concentration of 20 mg/mL was prepared with ethanol as solvent. The curcumin solution was dissolved in the sodium caseinate solution in a proportion of 1:10 . The bottom chloroform phase of the mixture was shifted and analyzed for absorbance at 419 nm (OD419) utilizing a UV-1800 spectrophotometer  after being stirred overnight at room temperature (22 °C) and after the mixture split into two phases. Based on the calibration curve created previously using standard solutions containing various concentrations of free curcumin dissolved in chloroform, OD419 was employed to calculate the curcumin concentration [w/v phosphotungstic acid). After a short period, the excess staining solution was removed from the grid. After the material had properly dried by air, a transmission electron microscope was used to analyze it.Zeta potential measurements  were used in order to assess the size of the nanoparticles (NPs). Transmission electron microscopy  was used in order to evaluate the size and morphology of the NPs in further detail. A diluted NP dispersion was adsorbed onto a carbon-coated Formvar film connected to a metal specimen grid. Blotting was used to remove the excess sample, and the grid was enveloped with a tiny drop of staining solution  with copper (Cu) Kα radiation was employed in order to determine the crystal structures of dried Cur-CS-NPs from the lyophilization of the Cur-CS-NPs dispersions. At 6°/min, 30 mA, and 40 kV, XRD data were received in the range of 2θ from 5° to 60°.The Cur-CS-NPs dispersions that were produced were stored for predetermined times at 4 °C , 25 °C , and 37 °C . The PDI, size, and zeta potential of the Cur-CS-NPs were measured.2), HFLS-RA cells and HFLS-OA cells were grown in a high-sugar Dulbecco’s modified eagle medium (DMEM) combined with 10% fetal bovine serum and 1% penicillin. At 37 °C, the cells were kept in humidified monolayer cultures in a 5% CO2 and 95% air environment. They were used in tests when the cell growth achieved around 70% confluence. Trypsin was used to break down the cell suspension, diluting the solution to the necessary concentration. For 24 h, the cells were injected onto 96-well plates (200 μL/well). Once the cells had attached to the wells and reached a confluency of about 70%, 20 μL of the drug at a specific amount was introduced into the wells. An amount of 10 μL of the CCK-8 reagent was introduced during the reaction’s first hour after the cells had been grown in the incubator for another 48 h. Using a Bio-Rad microplate reader  and a reference sample as a blank, absorbance at 450 nm was measured.In order to examine arthritis inhibition in vitro, the results of all the produced samples on the viability of HFLS-RA and HFLS-OA were investigated . In a ceAcontrol and Atreated are the absorbance of the untreated and treated cells, respectively.Cell viability was determined by the equation given below:4 cells/well) were seeded in 96-well plates and incubated for 48 h with Cur-CS-NPs and native curcumin  in the presence of tumor necrosis factor-α . Commercial ELISA kits  were used to quantify the levels of interleukin IL-8, IL-6, IL-1β, and MMP-1 in the cell supernatant as per the manufacturer’s guidelines [HFLS-RA and HFLS-OA cells (6 × 10idelines .5 cells/mL) were plated in 24-well Nunclon Delta cell culture plates  filled with RPMI 1640 media comprising 1% antibiotics and 10% heat-inactivated fetal bovine serum . The adherent cells were treated with curcumin and Cur-CS-NPs  after 6 h of incubation (37 °C/5% CO2), reconstituted in RPMI 1640 medium, and incubated for 24 h in a similar environment. The culture medium and non-adherent cells were isolated after 12, 24, 36, and 48 h, and the adherent cells were then rinsed three times with fresh water. The adhering cells were removed using a scraper. The obtained cells were dispersed in 10 mL of ethanol and processed with an ultrasonic cell pulverizer in order to isolate curcumin. Centrifugation  was used to isolate the pulverized cell residues and the extraction solution, and the supernatant was then applied to ultraperformance liquid chromatography mass spectrometry (UPLC-MS/MS)  in order to assess the intracellular curcumin. The UPLC-MS/MS analysis was carried out according to the procedure described by Wu et al. [m/z 367→149, the collision voltage and the cone voltage were set at 20 V and 60 V, respectively.HFLS-OA and HFLS-RA cells were used in a practical method to conduct curcumin cell uptake experiments. HFLS-OA cells or HFLS-RA cells ). Dunnett’s post-hoc test was used after a one-way analysis of variance  in order to identify statistically significant differences between the groups. We performed ultrasound treatment for different caseinate solutions and studied the influence of ultrasound treatment on the properties of the micelles. We prepared curcumin nanoparticles (curcumin NPs) by ultrasound treatment, which could change the size, zeta potential, surface free sulfhydryl group, and surface hydrophobicity of the casein micelles. Ultrasound treatment influenced the size, zeta potential, PDI, and encapsulation of curcumin . The aveCurcumin has been encapsulated into casein nanoparticles through several methods. The casein proteins self-assembled into spherical micelles with a mean size of about 166.3 nm. The curcumin molecules were attached to the casein micelles and produced complexes via hydrophobic bonding . Native-m/m). The high drug loading rate may lead to instability in the nano-system. In this study, the particle size of three nano-systems prepared with a casein concentration of 10 mg/mL and curcumin/casein ratios of 1:10 (m/m), 1.5:10 (m/m), and 2:10 (m/m) were evaluated. Changes in the nanoparticle size during storage at a cold temperature (4 ℃), normal temperature (25 ℃), and body temperature 37 ℃ were checked for the three samples of curcumin nanoparticles with different drug loads. The particle size of the newly prepared Cur-CS-NPs was 110–120 nm, but the size significantly increased at a point of time during storage (m/m) were relatively stable under cold storage, and the particle size did not change significantly until the 10th month of storage. At room temperature, the particle size of Cur-CS-NPs did not change significantly within 6 days, but changed significantly after more than 2 days at body temperature. The particle size of Cur-CS-NPs prepared with a curcumin/casein ratio of 1.5:10 (m/m) began to increase significantly after 4 months of cold storage and 6 days at room temperature, whereas the particle size enhanced significantly on the second day of storage at body temperature. The particle size of Cur-CS-NPs prepared with a ratio of curcumin/casein of 2:10 (m/m) changed significantly after 4 months of cold storage and 4 days of storage at room temperature, but increased significantly after 1 day of storage at body temperature. The lower curcumin/casein ratio yielded more stable Cur-CS-NPs. When actually used, Cur-CS-NPs are required to be stable for different periods of time. We can select the appropriate drug load rate to prepare Cur-CS-NPs. For use in food, Cur-CS-NPs should be stable for 8 months in cold storage, 6 days at room temperature, and 2 days at body temperature in order to meet the requirements for available storage and oral delivery.In the preparation of curcumin–casein nanoparticles (Cur-CS-NPs), a high encapsulation rate could be achieved as the caseinate solution was 10 mg/mL and the curcumin/casein ratio reached 2:10 . Native curcumin was obtained by dissolving curcumin in a mixed solution of polyethylene glycol (PEG300 (40%)-DMSO (10%)-Tween 80 (5%)-saline (45%) [Curcumin has anti-arthritic effects for patients with osteoarthritis (OA) and rheumatoid arthritis (RA) ,33. We mne (45%) . The nanCurcumin has a low bioavailability due to its low water solubility and crystallinity. The Cur-CS-NP dispersion overcame the problem of low water solubility. In order to demonstrate the effect of crystallinity, we performed XRD of Cur-CS-NPs and compared them to the pristine. In contrast to sodium caseinate, which had the typical amorphous XRD pattern, pristine curcumin displayed multiple unique peaks due to its crystallinity . The disArthritic cells can produce cytokines, namely IL-8, IL-1β, IL-6, and MMP-1, which are induced by TNF-α . In the Even in the early stages of the disease, inflammation is thought to play a role in the formation and progression of OA. Secreted inflammatory substances, including proinflammatory cytokines, are important mediators of the altered metabolism and increased catabolism of the joint tissue associated with OA. The primary proinflammatory cytokines thought to be associated with the pathophysiology of OA contain TNF-α, IL-1β, and IL-6 . In RA, In this work, curcumin was encapsulated in casein micelles in order to form curcumin–caseins nanoparticles using an appropriate duration of ultrasound treatment, which drove the hydrophobic groups to the inner micelles and the polar sulfydryl groups to the surface of the micelles. The obtained curcumin–caseins nanoparticles had an appropriate size (100–120 nm) for cellular uptake and excellent stability, and exhibited significantly higher inhibitory activity against the proliferation and proinflammatory cytokines for both HFLS-OA cells and HFLS-RA cells compared to native curcumin. This was due to better cellular uptake resulting from the low crystallinity of curcumin in nano-form and the appropriate nano-size. The work time of ultrasound treatment obtained in the present study is the referential value for the incorporation of different drug molecules, and the curcumin–caseins nanoparticle form has the potential to treat arthritis.


Correction to: Vis Comput Ind Biomed Art 5, 9 (2022)https://doi.org/10.1186/s42492-022-00105-4Following publication of the original article , the autThe CBAM” and “Results and discussion” due to negligence. The correct information is given below:In addition, the authors also identified some errors in the sections “W0.”“where σ represents the sigmoid function. Note that the MLP weights, W0 and W1, are shared for both inputs, and the rectifed linear unit (ReLU) activation function is followed by “The results of these experiments show that the Dice value for DCAU-Net reached 74.55% on the testing set. The basic U-Net network exhibited the lowest Dice of 46.29%, which is not suitable for segmenting aneurysm-containing images. The Dice value for U-Net with dense blocks was 7.95% higher compared with that for U-Net alone, proving the effectiveness of dense connections. The combination of U-Net and CBAM was less sensitive than the system that used only the SAM, but the Dice value was higher than that for U-Net combined with a single spatial or channel attention, indicating the effectiveness of the CBAM for segmenting aneurysm-containing regions. To validate the effectiveness of the MFB, a multi-scale fusion block was added to the improved U-Net up-sampling part. The Dice value increased by 1.63%, and the sensitivity increased by 5.03%, indicating that the MFB effectively improved the segmentation performance on aneurysm-containing images. The Dice value and the sensitivity of the traditional convolution block using the BN layer were, respectively, 8.43% and 10.52% lower than those of the currently proposed algorithm. This shows that the GN layer effectively improved the segmentation accuracy for aneurysm-containing images while reducing the memory space. The Dice value obtained using the RReLU function was 17.24% higher than that obtained using the ReLU function, proving the effectiveness of the RReLU function.”The original article  has been


With the outbreak and spread of COVID-19, a deep investigation of SARS-CoV-2 is urgent. Direct usage of this virus for scientific research could provide reliable results and authenticity. However, it is strictly constrained and unrealistic due to its high pathogenicity and infectiousness. Considering its biosafety, different systems and technologies have been employed in immunology and biomedical studies. In this study, phage display technology was used to construct a nonpathogenic model for COVID-19 research. The nucleocapsid protein of SARS-CoV-2 was fused with the M13 phage capsid p3 protein and expressed on the M13 phages. After validation of its successful expression, its potential as the standard for qPCR quantification and affinity with antibodies were confirmed, which may show the possibility of using this nonpathogenic bacteriophage to replace the pathogenic virus in scientific research concerning SARS-CoV-2. In addition, the model was used to develop a system for the classification and identification of different samples using ATR–FTIR, which may provide an idea for the development and evaluation of virus monitoring equipment in the future. With the outbreak and development of the COVID-19 pandemic since the end of 2019, the world is experiencing an ongoing unprecedented global health crisis. Because of the high pathogenicity and infectiousness of SARS-CoV-2 and the continuous emergence of coronavirus variants, it is more difficult to control the pandemic in a short time, implying that our battle against the COVID-19 pandemic remains long, arduous, and uncertain. Due to the high infectivity, pathogenicity, and instability of the virus, it is difficult to use the virus directly for scientific research. Therefore, multiple technologies have been proposed to synthesize a substitute in order to provide biosafety for scientific research and applications. For example, recombinant adenovirus type-5 (Ad5)- and type-9 (Ad9)-vectored COVID-19, which could express the spike glycoprotein of SARS-CoV-2, were produced and used for immune induction . FurtherOut of the many possible technologies available, phage display is a relatively mature technology that could allow the foreign protein to fuse into the phage capsid protein and display it on the surface ,7,8. TheRapid virus recognition will help ease the pandemic situation and benefit vaccine development. The widely used PCR and ELISA have some advantages towards the detection of the virus ,15,16,17E. coli. The M13 phage has five different capsid proteins, which are p3, p6, p7, p9, and p8. p7, p9, p3, and p6 are distributed on both ends of the phage in pairs, while p8 is wrapped on the surface in a spiral form [Considering the current research, we attempted to construct a nonpathogenic model by using the M13 phage to display the N protein of SARS-CoV-2. The M13 phage is a commonly used bacteriophage for phage display. It belongs to a kind of flexible filamentous virus and has host specificity for ral form ,30 , and sequencing , the infectivity of constructed phages decreased by 1–2 orders of magnitude. However, they still maintain the ability to infect bacteria as a virus.The results from the plaque assay are shown in 2 > 0.99 represents a good linear relationship for both standard curves in the two regression analyses. Compared with Model-N from the purchased standard plasmid  shifts over time (X-axis). For each line, the rising section describes the association process between Model-N and antibodies, while the descending section can explain the disassociation. In addition, the red smooth curves are the fitting results for the original experimental outcomes by software. 9 M−1, which may indicate a strong combination between Model-N and anti-N-protein antibodies.The reaction between Model-N and corresponding antibodies was monitored and recorded, as Through the above analyses, it could be proven that the N protein of SARS-CoV-2 has been expressed on the displayed phages of Model-N successfully. It has a relatively strong combination with anti-N-protein antibodies. Therefore, the constructed phage Model-N might possibly be used as a nonpathogenic model for immunology and biomedical research of SARS-CoV-2. On the one hand, the biological properties of the virus species are preserved and maintained; on the other hand, the properties of the N gene and N protein were both validated and confirmed on molecular and protein levels; the constructed model can probably be used as a biosafe substitute of SARS-CoV-2 for some research.This section attempts to provide a technical system for virus monitoring, classification, and identification based on the infrared spectral characteristics of Model-N and other relative materials. Specifically, since Model-N has been verified and confirmed at molecular and protein levels, we attempt to apply it in a spectrum study and utilize a PCA-LDA algorithm to provide a possible verification and performance evaluation idea for the development of real-time virus monitoring and early-warning equipment.Model-N, the M13 phage, the commercial SARS-CoV-2 N protein, and the BSA protein were selected for this study. ATR–FTIR was employed and data were collected using a thermo-fluorescence spectrometer .After data collection, principal component analysis (PCA) was performed for dimensionality reduction see . The cumAccording to the scatter plots, it can be seen that in the coordinate systems composed of PC1–PC2 and PC1–PC3 see , the fouIn summary, the prepared Model-N was applied in this section. Combined with ATR–FTIR and a PCA-LDA algorithm, the identification and classification of possible bio-threat substances were realized. This section is not only concerned with the application of the constructed model, but also provides an idea for the development and evaluation of virus monitoring equipment in the future. When new virus monitoring equipment needs to be simulated or tested, the prepared nonpathogenic virus-like model can be used as a reference/sample to evaluate the performance of the equipment without any contamination or hazards.9M−1, proving a strong interaction with anti-N-protein antibodies. We confirmed the possibility of using this nonpathogenic model instead of the pathogenic virus in COVID-19 studies. Furthermore, as a type of virus, the infectivity of the synthesized phages was maintained, and the biological properties of the virus species were preserved. Compared with the commercial N protein, the model is a kind of virus which may possess more biological properties akin to SARS-CoV-2. Therefore, the model we constructed might be a useful nonpathogenic substitute for SARS-CoV-2 for some research.In this study, we used phage display technology to fuse the nucleocapsid protein of SARS-CoV-2 to the M13 phage’s p3 protein. From the molecular study level, we verified the successful insertion of the target fragment; from the protein study level, we confirmed that the synthesized phages could express the N protein through Western blotting and ELISA. In addition, we described its applications differently. Firstly, we investigated the qPCR quantification and affinity interaction using the constructed model to imply the possibility of being a substitute in COVID-19 research. The standard curve was established successfully, and the affinity constant reached 1.229 × 10Furthermore, we employed the model in classification and identification of potential “bio-threats” with ATR–FTIR and algorithms. The results showed that the four relative samples, including the bio-threat Model-N, were classified well after PCA-LDA analysis and might be useful in developing and evaluating virus monitoring equipment in the future. Additionally, our research provides a new idea to study this highly pathogenic and infectious virus in a safe environment and confirms its feasibility.SfiI/NotI site for cloning the target gene [SfiI, NotI enzymes, and M13KO7 helper phages were purchased from New England Biolabs . E. coli TG1 and nuclease-free water were bought from Biomed . PrimeSTAR HS (Premix) for PCR amplification, T4 DNA ligase, and a DL5000 DNA Marker were purchased from TaKaRa . Agarose, kanamycin, chloramphenicol, BSA, PBS buffer, TBST buffer, TAE buffer, ELISA coating buffer, stop buffer, TMB single-component substrate solution, and SARS-CoV-2 N protein were bought from Solarbio . A PureYield™ Plasmid Miniprep System and a Wizard® SV Gel and PCR Clean-Up System were bought from Promega . Poly (ethylene glycol)  was bought from Sigma-Aldrich . Rabbit anti-SARS-CoV-2 N-protein polyclonal antibody and IRDye® 800CW goat anti-rabbit IgG (H + L) were bought from Bioss. Rabbit anti-M13 pAb-HRP was bought from Beijing Antaizhiyuan Technology Co., Ltd. . Skim milk powder was bought from Anchor. Syringe filter units (0.45 µm) were bought from Merck Millipore . A nitrobiocellulose filter membrane was bought from Bio-Rad . All broths and culture media were prepared in our lab. N gene standard plasmid (for qPCR) was bought from Shanghai Institute of Measurement and Testing Technology .The constructed phagemid vector pHB was donated by the Academy of Military Medical Sciences, and contained the chloramphenicol resistance gene, the plac promoter, the gIII gene of M13, and the get gene . SfiI, N® Imaging system was from LI-COR . A molecular interaction analyzer (ForteBio Octet K2) was bought from SARTORIUS . ATR–FTIR was bought from Thermo .A PCR amplifier was bought from Thermo Fisher . Electrophoresis was implemented by a Bio-Rad apparatus . An automatic microplate reader (SPARK) was bought from TECAN . An OdysseySfiI/NotI restriction digest sites at both ends. Additionally, the corresponding primers were designed and synthesized according to the redesigned target genes . Then, PCR was employed in a 50 μL reaction system, which contained 25 μL PrimeSTAR HS (Premix), 50 pmol/μL primers for both ends, 1 μL template plasmid, and 23 μL nuclease-free water. The PCR amplification reaction program was set at 95 °C/10 min for initial denaturation, then followed by 30 cycles at 94 °C/1 min for denaturation, 56 °C/1 min for annealing, 72 °C/90 s for extension, and another 72 °C/10 min for a final extension. After purification, the obtained genes were digested with SfiI and NotI, sequentially. Because of the different reaction temperatures and buffer environments for the two enzymes, a step-by-step procedure was required. The compositions are displayed in The gene corresponding to the N protein of SARS-CoV-2 was investigated at the National Center for Biotechnology Information (NCBI website/Gene ID: 43740575). The original sequence was redesigned and synthesized, including the addition of The produced fragments from double enzyme digestion were purified and named prepared-N-gene.SfiI and NotI, employing conditions similar to mentioned above. Following purification and recovery after digestion, the vector was named prepared-pHB and prepared for construction.Similarly, the phagemid vector pHB-1HSCFV was digested by the same enzymes After the preparations of inserted fragments and vectors, the recombinant vectors were constructed following the conditions in E. coli TG1 cells. Then, 100 μL TG1 was inoculated in 5 mL of 2xTY broth with chloramphenicol (34 mg/mL) and cultivated overnight at 37 °C with a shake rate of 250 rpm. The following day, the cultivated mixture was inoculated into 40 mL of 2xTY broth at a ratio of 1:100 and cultivated at 37 °C to its logarithmic growth phase while shaking. Then, the M13KO7 helper phages were added with a titer of1 × 109–4 × 109 pfu/mL. The mixture was cultivated at 37 °C at 150 rpm for 2 h. After centrifuging at 4000× g for 10 min, the precipitates were resuspended in 200 mL of 2xTY broth containing kanamycin (25 mg/mL). The culture mixture was cultivated overnight at 30 °C at 250 rpm. Then, after centrifugation (4000× g/4 °C/10 min), 20% polyethylene glycol 8000/2.5 M NaCl was added to the supernatant and shaken violently. Another centrifugation (9000× g/4 °C/20 min) was employed after a one-hour incubation on ice, and the pellet was resuspended with 2–3 mL of PBS and filtered through a 0.45 μm filter membrane. The constructed products were stored at 4 °C.The recombinant pHB-N was transformed into the competent SfiI/NotI), and sequencing to verify the insertion of the target gene. The electrophoresis results were used to determine whether the target genes were inserted or not.The constructed pHB-N was identified by PCR amplification, double digestion (® 800CW goat anti-Rabbit IgG (H + L) was employed for secondary antibody incubation. Followed by washing 4 times with TBST, color development was performed and observed by the Odyssey® Imaging system.Western blotting was employed to detect whether the N protein of SARS-CoV-2 was expressed on the synthesized phage display. Western blotting, also known as immunoblotting, is a method of using blotting technology to transfer proteins to membranes and then detect particular proteins in complex samples according to the specific binding of antigens and antibodies. Firstly, the mixed protein samples were separated by polyacrylamide gel electrophoresis (PAGE), and then blotted onto a solid-phase medium (such as a nitrocellulose (NC) membrane). Then, the protein or polypeptide on the solid-phase carrier was used as an antigen, which may have specific binds with the primary antibody. After that, the mixture was incubated with the enzyme or isotope-labeled secondary antibody. The target protein could be observed by substrate color development or autoradiography. Regular steps of Western blotting were applied. First of all, electrophoresis was accomplished with the Bio-Rad apparatus. Then, membrane transfer with a 0.45 μm NC filter membrane was achieved, followed by blocking with 5% skim milk powder/BSA for 1 h at room temperature. Then, rabbit anti-SARS-CoV-2 N-protein polyclonal antibody was used for primary antibody incubation and IRDyeSandwich ELISA was used to detect if there was any sign of the expression of N proteins of displayed phages. A 96-well plate was coated with rabbit anti-SARS-CoV-2 N-protein polyclonal antibody. After washing and blocking, the displayed phages with different diluted ratios were added for binding. Then, the rabbit anti-M13 pAb-HRP was added and combined to form a sandwich structure. After adding TMB substrate, the OD values at 450 nm were obtained by the automatic microplate reader.E. coli TG1, and the mixture was placed on LB solid culture media (with kanamycin). The cultures were cultivated overnight at 37 °C, and all formed plaques were counted the next day.The gold standard method for titer determination, plaque assay, was employed to determine the titer of the synthesized phages. The displayed phages with different dilution ratios were used to infect Real-time qPCR standard curves were established for the purchased standard plasmid containing the N gene and the recombinant plasmid pHB-N of the construction model see . The posThe molecular interaction instrument Fortebio Octet K2 was used to determine the affinity of the expressed N protein on the synthesized phage and its antibody ,36. The Model-N, as a synthetic phage, was designed to display the N protein of SARS-CoV-2. It could be used as a potential bio-threat when developing or evaluating the performance of real-time virus monitoring and early-warning equipment. In this section, Model-N, the M13 phage, the commercial SARS-CoV-2 N protein, and the BSA protein were selected to employ ATR–FTIR (using a thermo-fluorescence spectrometer). The data were collected and processed with the PCA-LDA algorithm to realize the classification and recognition of the four samples. All data were analyzed by IBM SPSS Statistics 26.0.


A self-powered and highly sensitive tribo-label-sensor is proposed as the substitution of infrared sensor for addressing current issues in label printer.Tribo-label-sensor shows higher integrability, sensitivity, reliability and universality than traditional infrared sensor for label identification, positioning and counting.The online version contains supplementary material available at 10.1007/s40820-022-00999-y. Label-sensor is an essential component of the label printer which is becoming a most significant tool for the development of Internet of Things (IoT). However, some drawbacks of the traditional infrared label-sensor make the printer fail to realize the high-speed recognition of labels as well as stable printing. Herein, we propose a self-powered and highly sensitive tribo-label-sensor (TLS) for accurate label identification, positioning and counting by embedding triboelectric nanogenerator into the indispensable roller structure of a label printer. The sensing mechanism, device parameters and deep comparison with infrared sensor are systematically studied both in theory and experiment. As the results, TLS delivers 6 times higher signal magnitude than traditional one. Moreover, TLS is immune to label jitter and temperature variation during fast printing and can also be used for transparent label directly and shows long-term robustness. This work may provide an alternative toolkit with outstanding advantages to improve current label printer and further promote the development of IoT.The online version contains supplementary material available at 10.1007/s40820-022-00999-y. Duri. fields , 7, lase. fields , capacit. fields  and ultr. fields  sensor. . fields . However., with the highest motion resolution in nanometer [Recently, originating from mechano-induced Maxwell displacement current –15, tribanometer . HarnessIn this work, we propose a self-powered, highly sensitive and embedded tribo-label-sensor (TLS) for the fast and stable label printing. Based on the mechanism of contact-separation (C–S) mode TENG, TLS is designed on the intrinsic roller structure inside the label printer without changing the overall structure, which is a universal strategy for various label-printers. In the experiment, device parameters on sensing performance and deep comparison with infrared sensor are systematically investigated. As the results, TLS delivers 6 times higher signal magnitude than traditional one and is immune to label jitter and temperature variation during fast printing. Moreover, TLS can also be used for transparent label directly and keeps stable sensing performance after long-term operation. Additionally, a virtual-based software platform is successfully developed for demonstrating the potential capability of TLS in practical label printing. This reported TLS may have great potential to address some hardships in current label printer and further promote the development of IoT.The potential distribution was numerically calculated using a commercial software COMSOL Multiphysics (5.6a version). The electrostatic field module was used to obtain all potential distributions at different separation distances under a 2D model.. Two aluminum rods of length 50 mm and diameter 6 mm were machined by lathe  to form step shafts of length 5 mm and diameter 3 mm at the ends of each. Subsequently, one of the aluminum rods was covered with tribo-material. Finally, bearings were installed at the ends of each aluminum rod and connected to the external frame by preload of springs.The detailed fabrication process of TLS is shown in Fig. S1−1 driven by a linear motor . A fan was used to trigger random jitter of the label. Heat gun (GJ-8018LCD) was applied to heat the sensor surface and thermal imaging camera  recorded the real-time temperature. The virtual label-printing interface was constructed based on LabVIEW, which could realize real-time data acquisition control, analysis and label printout display.A field-emission scanning electron microscope (Hitachi SU8010) was used to characterize the surface morphologies of FEP film. The electrical signals were recorded using a programmable electrometer (Keithley 6514). The operation speed of the label is 0.1 m sVOC) and short-circuit current (ISC) output of TLS in one sensing period. As the printer operates continuously, state I and II revolve, generating periodical signals  and label width W2 (15 mm), the sensing signal of TLS is comparable to that of infrared sensor, even with higher magnitude.For any label-printer, the basic label-printing processes and main components are schematically illustrated in Fig. als Fig. f. ExcitiW1) and the width of label (W2) are two basic label specifications, which need to be detected by the label-sensor. Figure VOC signal of TLS for a typical sensing process. It can be seen that the half-height time-width t1 of the pulse relates to the label-interval W1 and the label-width W2 is reflected by the time-gap t2 between adjacent pulse signals, respectively, which are determined by the sensing mechanism of TLS. Therefore, with label-interval W1 changing, the time-width t1 varies accordingly, as VOC signal plotted in Fig. ISC signal shown in Fig. S4. It is worth noting that the missing of labels can also be further analyzed from the time-width t1 . Figure t1, t2 and W1, W2, t1 shows excellent linearity to W1 (from 2 to 4 mm), and t2 keeps constant with the fixed W2 (15 mm). In addition, the output signal of infrared sensor for sensing the labels with different specifications is recorded in Fig. S6, which indicates that TLS holds the same sensing characteristic with the traditional one. Nevertheless, for the same W1, t1 measured by TLS is always smaller than that of infrared sensor , and can detect transparent label directly. The fidelity and sensitivity of TLS could be further improved by roller parameter and tribo-layer optimization. Moreover, the mechanical jitter and generated heat during continuously fast printing showed no interference to the sensing signal of TLS. To demonstrate the application feasibility, TLS maintained both excellent electric and mechanical durability after 30,000 operation cycles. The potential capability of TLS in practical label printing was also successfully realized using a virtual-based software platform. The superior advantages to infrared sensor in self-powered, integrability, sensitivity, antijamming and universality clearly present TLS as an outstanding alternative toolkit to further promoting label printer technology and IoT.Supplementary file1 (MP4 22456 KB)Supplementary file2 (MP4 15329 KB)Supplementary file3 (PDF 1241 KB)Below is the link to the electronic supplementary material.


Symptoms of mental stress are a hallmark of the COVID-19 pandemic. We hypothesized that just testing for COVID-19 could act as an effective stressor for persisting symptoms of mental distress including posttraumatic stress disorder. Our study aimed to determine whether personal beliefs on individual control and competence  correlate with symptoms of mental distress and positive screening for post-traumatic stress disorder during a 9-month observational period.Between March and December 2021, we applied online versions of the Questionnaire on Competence and Control Expectations (FKK), the Depression, Anxiety, and Stress Score (DASS), the Short Screening Scale for DSM-IV Posttraumatic Stress Disorder (PTSD), and a medical history questionnaire for COVID-19 symptoms (visit 1). 48 hours after negative COVID-19 testing, DASS was repeated to address relief effects on mental distress (visit 2). Following 90 days (visit 3), development of mental distress was addressed by a combination of DASS and PTSD, while the possible long-term manifestation of PTSD was evaluated 9 months later (visit 4).n = 867) demonstrated a positive screening for PTSD, while after nine months (at visit 4), 8.9 percent of the remaining sample (n = 204) had positive screening results. The mean age was 36.2 years; 60.8% were women, 39.2% men. In contrast to individuals with negative PTSD screening, these participants demonstrated a significantly different LoC personality profile. This was confirmed by the results of both DASS and the COVID-19 medical history questionnaire.At visit 1, 7.4 percent of the total sample (Following testing for COVID-19, individuals with positive long-term PTSD screening present with significantly different personality traits than those w/o suggesting that self-confidence and effective control over one’s own actions serve as a protective function against mental distress. In lineIndividual personality traits based on domains of competence and control beliefs (LoC) correlate with positive screening results for PTSD.(1).What is the influence of domains of competence and control beliefs on mental health factors assessed using the standardized questionnaire ‘depression, anxiety and stress score’ (DASS)?(2).Does a negative COVID-19 test result relate to development of anxiety, depression, and stress as assessed by DASS?(3).Do people experiencing symptoms of COVID-19 and people who are subjectively symptom-free at visit 1, differ significantly concerning their DASS results?(4).Is there a significant change in personal depression, anxiety, or stress experience between the three visits that DASS is applied?(5).Do the results of PTSD screening at visit 1 differ significantly between clients subjectively experiencing COVID-19 symptoms and those who do not?We conducted a prospective questionnaire survey addressing the influence of personality traits on the development of stress, anxiety, depression, and posttraumatic stress disorder (PTSD) after COVID-19 testing. The study protocol was approved by the Ethics Committee of the Medical University of Vienna (EK1535/2020). The following validated inventories were used:(a)Competency and Control Beliefs Questionnaire (FKK) ;(b)Depression Anxiety Stress Scales (DASS) ; and(c)German version of the Short Screening Scale for DSM-IV PTSD .At study begin, the third general lockdown had just ended and the rate of COVID-19-associated deaths in Austria was close to 10,000 . The perAfter written informed consent, all test persons underwent pseudonymization and received a link via mail on their mobile phones. This approach provided the opportunity to complete the initial questionnaires during visit 1 at the test site while still waiting for the COVID-19 test results. Only fully completed inventories were evaluated; likewise, further participation in the survey was only possible if all inventories were completed. In case of missing answers, a reminder email was sent after 24 h.At visit 1 (day 0), three validated tests  and a medical history questionnaire addressing the major symptoms of COVID-19 infection see  were admAt visit 2 (two days after visit 1), DASS was applied. At visit 3 (90 days after visit 1), both DASS and the Short Screening Scale for PTSD were provided.For the final investigation at visit 4 (270 days after visit 1), only the Short Screening Scale for PTSD was used.Validated questionnaires:Questionnaire on Competence and Control Beliefs (FKK) .The FKK represents an enhancement of Rotter’s social learning theory developed by G. Krampen. The questionnaire records generalized expectations with regard to scope for action; these expectations and attitudes relate to personal experience, learned competence and the subjective evaluation of the given situation . It can four primary scales: (a) - (d); two secondary scales: (e) - (f); and one tertiary scale (g). These capture the following personality traits:The seven scales of the FKK include (a)Self-concept of one’s own abilities (FKK-SK) in the sense of recording possibilities for action against the background of self-confidence (Cronbach α = 0.72-0.82),(b)Internality in the sense of having the power to determine one’s own life ,(c)Social externality or “powerful others control” as an expression of the dependence of an individual on the social environment , and(d)Fatalistic externality or “chance control” (FKK-C) as a measure of the external determination of one’s own convictions (Cronbach α = 0.75-0.81).The two secondary scales are calculated as follows:(e)Self-efficacy = sum SK + I (FKK-SKI) and(f)Externality = sum P + C (FKK-PC),The tertiary scale is calculated as follows:(g)Internality vs. externality (SK + I) - (P + C) (FKK-SKI-PC) serve as a criterion of self-efficacy without personal dependency vs. helplessness and external determination.Depression-Anxiety and Stress-Score (DASS) , German Short Screening Scale for PTSD . The scale includes nine validated items on thought and emotion avoidance consisting of: loss of interest, sense of alienation, numbness/deafness, unfulfilled plans for the future, sleep disturbances, distressing memories and distressing dreams/nightmares. Cut off value was ≥ 4. The procedure has a high internal consistency (α = 0.90), validity, and economy . Thus, iThe test procedure allows for an additional self-categorization of mental traumata. In our study, five categorized traumata could be discerned: No trauma, anxiety, isolation, feeling of loss, and illness.A categorized medical history questionnaire was used to collect the sociodemographic characteristics of the participants see  and indiAll data were pseudonymized to avoid inference to individual persons; sensitive data are stored access-protected on the server of the MUW. ICFs are only accessible to authorized persons. After completion of the study, data were archived in the data management system according to legal requirements. Participants did not face any risk or individual benefit.2 .Among the subjectively perceptible symptoms frequently reported during COVID-19 infection are symptoms of respiratory tract infection, such as fever, cough, and shortness of breath, as well as sore throat, head and muscle pain, eye pain, and diarrhea see . As a ren = 914), 47 (5.1%) PTSD questionnaires were not or only partially completed and thus not further processed. Of the remaining 867 PTSD questionnaires, 64  had a positive screening for PTSD (Of the questionnaires returned at the start of study (for PTSD . At day 2 ≥ 0.01), so that the two main effects had to be interpreted differentially post hoc. The results for all three scales of the DASS showed significantly higher scores for PTSD (p’s < 0.001), each with significant effects for depression (η2 = 0.26), anxiety (η2 = 0.26) and stress (η2 = 0.19). The increase in depression scores for those with PTSD over 90 days was steady but not significant . Similarly, a non-significant increase was observed for anxiety  and stress .The change in depression, anxiety and stress was tested on the basis of the DASS surveys  on day 0The trajectories of depressive states, anxiety and stress for participants with and without PTSD symptoms are illustrated in n = 782). p ≤ 0.007) including the differences between PTSD groups with small to moderate effect sizes (d between 0.36 (for FFK-I) and 0.79 (for FKK-SKI-PC).When comparing the personality profiles according to FKK with the PTSD scores at baseline, a significantly different personality profile was observed between individuals with (n = 62) and without positive PTSD screening (n = 844 protocols for visit 1 (day 0), the predictability of the criterion occurrence of PTSD was tested using binary logistic regression on the basis of the 4 FKK primary scales SK, I, P, C. The model fit was assumed using the non-significant Hosmer-Lemeshow test, p = 0.466. The model fit could be assumed based on the non-significant Hosmer-Lemeshow test, p = 0.461. By means of stepwise backward selection, FKK-SK  remained as a protective factor and FFK-C  as a risk factor with significant explanatory value for the occurrence of PTSD in the last model step. The coefficient of determination for the explained proportion of variance according to Nagelkerke’s R2 reached 10.4%.On the basis of 2adj. reached 30.5% for depressive states, 23.8% for stress and 28.6% for anxiety.Similarly, in order to assess the explanatory value of the four primary FKK domains FKK-SK, FKK-I, FKK-P, FKK-C for the three DASS criteria, multiple linear regressions were performed based on 872 cases. By means of stepwise backward selection, the predictors for the prognosis of depressive states, stress and anxiety were used accordingly. The results suggest that FKK-I should be excluded as a non-significant predictor for prognosis of depressive states, stress, and anxiety. For prediction of depressive states, the predictors FKK-SK (β = −0.30), FKK-P (β = 0.21) and FKK-C (β = 0.14) have small to moderate effect sizes. The same is true for prediction of stress using FKK-SK (β = −0.26), FKK-P (β = 0.22) and FKK-C (β = 0.09) as predictors. For anxiety, FKK-SK (β = −0.32), FKK-P (β = 0.22) and FKK-C (β = 0.10) each have a comparable predictive power (p’s < 0.01). The explained variance ratio RThe present long-term study took place from mid-March 2021 until December 2021 at the Wiener Stadthalle, the largest COVID-19 test site in Vienna, shortly after the advent of the α-variant of the SARS CoV-2 virus in Austria. This was a time characterized by widespread media coverage of rising morbidity and mortality rates in Austria. Since November 2020, it had been mandatory to wear FFP2 masks at any location outside the immediate living areas. Frequent, even daily, testing for COVID-19 by nasal swabs was obligatory to participate in professional and social life. Presuming that (a) COVID-19 testing, independent of its outcome, might act as a trigger for psychological stress, and (b) following the hypothesis that individual beliefs about self-competence and action control would significantly contribute to this kind of negative stress, randomly chosen individuals with negative COVID-19 test results were invited to participate in the study.Locus of Control (LoC) is considered central to individual personality and as such, part of the theory of metacognition –30. OngoThe study addressed the questions whether personality traits according to Rotter’s Locus of Control (LoC) would correlate with (a) symptoms indicative of stress, anxiety, and depression, and (b) with a positive screening for post-traumatic stress disorder (PTSD). To this end, we applied the advanced questionnaire on competence and control beliefs (FKK) by Krampen et al. , 33.p = 0.001, OR = 0.92, 95%-KI ) can be regarded as a protective factor and the FFK-C scale  as a risk factor for the criterion occurrence of PTSD. The results of our study indicate that personality traits favoring low self-confidence and high externality could act as a precondition for the development of a post-traumatic stress disorder in the COVID-19 pandemic. This state of mind has already been encountered during the pandemic  as a ripandemic , likely pandemic .p < 0.001). Given the tendency of PTSD to stabilize over longer time intervals, it may be noteworthy that all DASS scores increase in PTSD screening positive individuals, in particular the scores for depression and anxiety, albeit not significantly  and 50 women (9%) of the total sample described PTSD symptomatology at study entry. Trauma defines that the affected person experiences a threatening situation, which is assessed as vital threatening and is accompanied by the feeling of helplessness and being at the mercy of the situation, as well as the shaking of the self and world view. Statistically, two-thirds of the world’s population experience trauma in their lifetime, with one-third developing PTSD and two-thirds coping with trauma through their own experiential and competence mechanisms and social systems . TraumatThe main weakness of this study is our lack of knowledge if psychological or psychiatric help in any form had been used by the participants. Furthermore, the cut-off scoring procedure in the DSM IV screening test for PTSD may not correspond to the latest DSM V convention. In addition, it was not possible to assess the influence of COVID-19 coverage in the media on the mental state of the participants.The original contributions presented in this study are included in this article/The studies involving human participants were reviewed and approved by Ethics Committee of the Medical University of Vienna, Approval No. 1535/2020. The patients/participants provided their written informed consent to participate in this study.All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.


The one-, three-, and five-year overall survival rates were not significantly different between the two groups (p = 0.056). In univariate analysis, a high pre-LT NLR was associated with poorer survival in the high MELD group . NLR cut-off values of ≥10.7 and <10.7 were predictive of mortality, with an AUC of 0.705 (90% CI: 0.532–0.879). The one-, three-, and five-year post-LT survival rates were significantly higher among the recipients with an NLR < 10.7 than those with an NLR ≥ 10.7 (p = 0.005). Conclusions: Pre-LT MELD score ≥ 20 was associated with a higher risk of developing post-LT septic shock and mortality. The pre-LT serum NLR is a useful predictive factor for clinical outcomes in patients with HCC with high MELD scores.Background: Patients with hepatocellular carcinoma (HCC) tend to be referred for liver transplantation (LT) at an early stage of cirrhosis, with lower pre-LT Model of End-Stage Liver Disease (MELD) scores. We investigated the impact of high MELD scores on post-LT outcomes in patients with HCC and validated the prognostic significance of the neutrophil-to-lymphocyte ratio (NLR). Patients and Method: This retrospective single-center cohort study enrolled 230 patients with HCC who underwent LDLT from 2004–2019 in our institute. We defined a high MELD score as ≥20. Results: The MELD < 20 and MELD ≥ 20 groups comprised 205 and 25 cases, respectively. Although there was no significant difference in disease-free survival between the two groups ( Since the introduction of Milan’s criteria , the lonAlthough some studies have reported that high MELD scores do not adversely affect patient or graft outcomes ,6, recipThe neutrophil-to-lymphocyte ratio (NLR), a peripheral inflammatory parameter, has been shown to be useful in predicting graft dysfunction, acute rejection, and high-risk explant features ,13,14,15This was a retrospective single-center cohort study. We screened 746 adults who underwent LDLT between October 2004 and October 2019 at the Chang Gung Memorial Hospital, Linkou, Taiwan. We excluded patients without HCC or those who had an insufficient follow-up period to monitor the outcome. Ultimately, 230 HCC recipients with different levels of end-stage liver disease were enrolled. We followed the University of California San Francisco (UCSF) criteria (solitary tumor ≤ 6.5 cm or up to three tumors < 4.5 cm) for patient selection [The pre-operative preparation and LDLT techniques were executed in accordance with our institutional protocol ,19. In gPost-LT immunosuppressant administration was introduced in our previous publications , in whicAlpha-fetoprotein (AFP) and Doppler ultrasound were performed for tumor surveillance every 3 months after LT, and computed tomography was conducted every 6 months or when necessary. HCC recurrence after LT was defined as a tumor found in any location throughout the body following a period when it was undetectable. The disease-free survival (DFS) was calculated from LT to the day of tumor recurrence.The primary outcome was long-term overall survival (OS), which was calculated from the transplantation date to the date of death; data from patients who were alive as of 31 June 2021 were censored. Several types of infection were considered in our study. Post-LT infection, defined as any culture evidence of invasion and growth of pathogens  in recipients. Detectable cytomegalovirus (CMV) antigen or CMV DNA in the patient’s blood was a prerequisite for CMV disease in the presence of clinical symptoms or signs . Septic p-value of <0.100 were subsequently entered into the multivariate analysis. The Kaplan–Meier method with a log-rank test was used to compare patient survival between the groups. All statistical analyses were performed using IBM SPSS® , and two-tailed p-values of <0.05 were considered statistically significant. Receiver operating characteristic (ROC) curve analysis was performed to determine the predictive value of a clinical factor, and the optimal cut-off point was determined using the Youden index to assess the predictive accuracy. Linear regression analysis was used to assess the relationship between the MELD score and serum NLR.Continuous variables are reported as median values and mean values ± standard deviations, with a range of minimum and maximum ranges, while categorical variables are reported as numbers (percentages). Pearson’s chi-square test, independent t-test, and nonparametric methods were used to compare clinical parameters. The Cox proportional hazard risk model was the choice of time-to-event outcome regarding OS to reveal the effect of each parameter, and a binary logistic regression model was used to calculate the relationship between several risks and dichotomous events (the presence and absence of post-LT septic shock). In the univariate analysis, all potential parameters were included, and only factors with a n = 186, 80.9%) and those with an etiology of viral hepatitis . The mean Child-Pugh and MELD scores were 7.7 ± 2.2  and 12.9 ± 5.7 , respectively. Among them, 189 (82.2%) patients received locoregional therapy targeting HCC at least once before LT. Two hundred and eighteen (94.8%) recipients obtained a right liver graft, with a mean GRWR of 0.95 ± 0.18% . Explant histology was examined by a certified hepatopathologist, and the findings showed that 63 (27.4%) patients had tumor status beyond the UCSF criteria. The mean size of the maximum tumor and the summation of all tumors were 2.7 ± 1.5 cm  and 4.9 ± 3.6 cm , respectively. The mean tumor number was 2.7 ± 2.7, and the median for the given data was 2.After reviewing all of the recorded data, the demographic and laboratory characteristics of all 230 patients are summarized below . The meaWe investigated independent pre-operative factors to predict post-LT long-term survival . Our resp < 0.001).Subsequent multivariate analysis revealed a predominant weight of septic shock in survival prediction . Post-LTn = 10/18, 55.6%) and 2 high-MELD patients  with septic shock were diagnosed with pneumonia, which seemed to have a great significance in developing this lethal situation.The leading cause of post-LT infection was a blood-stream infection or catheter-associated infection, followed by similar proportions of intra-abdominal infection and pneumonia. Eighteen and six patients experienced septic shock in the low and the high MELD group, respectively. Besides this, over half of low-MELD patients with septic shock , confirming that the pre-transplant condition and intra-operative course were more complicated in the high MELD score group. Most oncological factors  were not significantly different between the MELD < 20 and MELD ≥ 20 groups. Notably, there were fewer patients who received locoregional treatment for HCC before LT in the high MELD group . Details were accessed according to different intentions of locoregional treatment before LT, and there were 11 (5.4%) and 1 (4.0%) for curative intention, 143 (69.8%) and 11 (44.0%) for bridge therapy before LT, 21 (10.2%) and 2 (8.0%) for tumor down-staging in the low and the high MELD group, respectively. Regarding the proportions of those receiving down-staging therapy, there was no significant difference in the two groups (p = 0.801). Most of the recipients had TACE-based locoregional therapy, except seven patients in the low MELD group and 1 case in the high MELD group, who received radiofrequency ablation only. Concerning the observation time from the latest locoregional treatment to the time of transplantation, there were 88 of the 175 (50.3%) and 9 of the 14 (64.3%) patients who received LRT with at least a 3 month period in the low and the high MELD group, respectively (p = 0.313).A comparison of demographic data between the high (≥20) and low (<20) MELD score groups is summarized in p = 0.629). Over half of the mortalities were infection-related, followed by HCC recurrence. Regarding infections, CMV disease (p = 0.007), severe CMV disease (p = 0.028), and septic shock (p = 0.019) were significantly more prevalent among the high MELD group. As a result, the OS was worse in the high MELD score group; the 1-, 3-, and 5-year OS rates were 85.8%, 77.5%, 71.1% for patients in the low MELD group, and 72.0%, 56.0%, 52.0% for patients in the high MELD group, respectively (p = 0.056). These results support the view that pre-transplant immunity was indeed more compromised in the high MELD group, although this weakened condition did not lead to significant tumor recurrence.Next, we examined whether high MELD scores had a negative impact on post-transplant outcomes. Tumor recurrence, acute rejection, and in-hospital infections were considered to be associated with post-LT survival and were compared between the two groups. The mean follow-up period of the present study was 83.65 ± 52.80 months . p = 0.025, HR: 3.28, 90% CI: 1.37–7.84). We confirmed that a high MELD score is an independent risk factor for post-LT septic shock, which could lead to poor prognosis.In p = 0.027, HR: 1.13, 90% CI: 1.02–1.27), but also compromised post-LT survival .We further investigated negative predictors in the high-risk subgroup. Univariate analysis was performed for pre-operative factors to predict septic shock and survival using the Cox regression model in patients with HCC with a high MELD score (≥20) receiving LDLT . The folp = 0.005; ROC analyses were performed to quantify pre-transplant serum NLR among recipients with a high MELD score to determine the optimal cut-off thresholds to predict the two main clinical outcomes . The Concordance index (C-index) of NLR in predicting mortality was 0.788  . Poor ouInteractions between NLR and post-LT outcomes in patients are likely to be complex. A high NLR represents a combination of neutrophilia and lymphocytopenia, and the former may indicate an active or unsolved infection, while the latter may imply insufficient host immunity and anti-cancerous ability ,31,32. MDespite a moderate correlation between the MELD score and serum NLR, which has been engaged in a predictive capacity for HCC recurrence , our stuThis study has several limitations in addition to the retrospective design. First, a type II error may exist because of the relatively small sample size. Second, this research was conducted based on laboratory information at a single time point before LT surgery, rather than the average from peri-operative time frames, and as such, a lack of dynamic evaluation and bias may arise. Third, direct evaluation of independent pre-operative risk factors for survival is beyond the preset accessibility. We focused on septic shock and its impact on post-transplant outcomes. Therefore, larger prospective studies are needed to validate our findings. In conclusion, managing high MELD scores in recipients with HCC remains a challenge. Although the DFS is not worse in these patients, they are more vulnerable to severe infections and develop poorer outcomes. A high pre-LT NLR can be used as a negative predictor of clinical outcomes in this high-risk subgroup. Early recognition of high-risk patients and implementation of prophylactic management are mandatory to prevent post-LT complications and optimize the overall outcomes after LDLT.